The T cell response to protein antigens depends on antigen processing and presentation by the antigen presenting cell. Proteins are partially degraded into antigenic fragments by one of two processing pathways, and the peptides bind the MHC groove and are transported to the cell surface for presentation to T cells. In this project, we will use normal intracellular traffic signals to manipulate protein entry into each processing pathway and the subsequent transport of processed peptides for MHC binding. For soluble antigens entering the cell by endocytosis, we are studying the effect of adding or deleting a fusion peptide from the amino end. Proteins capable of membrane fusion may enter the cytoplasmic processing pathway directly, without exposure to endosomal proteases, leading to presentation with MHC class I. For endogenously synthesized proteins, we are studying the effect of the endoplasmic reticulum (ER) retention signal KDEL added to or deleted from the carboxyl end. Proteins retained in the ER may be blocked from entering the cytoplasmic processing pathway and may no longer yield peptides for presentation with MHC class I or II. This would establish the site of entry into this pathway as distal to the ER and would also suggest ways to protect foreign proteins from immune recognition. So far, we have prepared plasmid DNA coding for all these protein constructs under control of a T7 RNA polymerase promoter, and we are now testing them for protein expression when co-infected with vaccinia virus expressing the polymerase. If the protein distribution obeys the new signals, we will test each construct for the ability to stimulate T cells specific for the products of one processing pathway or the other.